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Herein, to achieve individual and concomitant quantifications of phospholipid classes, an absolute quantification 31P NMR method using an internal standard was examined. Phospholipid standards and dietary foods were dispersed to prepare test solutions in an anionic surfactant (sodium cholate) solution containing EDTA, as a modification based on a reported method. Each phospholipid class showed a reproducible chemical shift at a near-neutral test solution pH of 6.90±0.04 and temperature of 30.0±0.1°C. The quantity of synthetic phosphatidylcholine measured using 31P NMR was consistent with that measured by 1H NMR using an internal standard. As the principal phospholipid class of soybean and egg yolk lecithin is phosphatidylcholine, the measurement conditions of 31P NMR (pulse interval time and number of scans) were optimized such that minor phospholipids, including lysophospholipids, also present in lecithin could be quantified simultaneously. Phospholipid classes in commercial polar lipid samples derived from porcine brain, yeast, and soybean were individually quantified using the above conditions. Using phosphoserine as the internal standard material allowed the absolute molar quantity of the phospholipid class to be precisely determined with traceability to the SI. The determined molar amounts of phospholipid classes were then translated to the weight amount by assuming that each phospholipid class contained two stearic acid molecules as the constituent fatty acid. The calculated total contents of each phospholipid class by 31P NMR were in good agreement with those obtained by molybdenum blue colorimetry. Furthermore, the quantitative values of the principal phospholipid classes in the polar lipid samples obtained by 31P NMR corresponded in a broad view, however, was more informative for the separation of individual phospholipid species rather than the quantitative 2D thin-layer chromatography.

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The pharmaceutical industry as well as European and US governing agencies have indicated the need for more accurate, high resolution, characterization of complex drug materials, nanomedicines, to facilitate their development and eventual approval. In particular, accurately measuring the size, zeta-potential, and concentration of nanomedicines is desired. Herein we demonstrate the comprehensive and high resolution analysis capabilities of tunable resistive pulse sensing (TRPS) on the most widely approved nanomedicines to-date, liposomal particles. The number-based size distribution, concentration and volume fraction of liposomes formed by extrusion through a 100 nm or 200 nm Nucleopore filter membrane are shown as well as how freeze-thaw aggregation changes individual liposomes and the overall size distribution. In addition, the simultaneous size and zeta-potential analysis capabilities of TRPS is used to characterize the homogeneity and difference between liposomes made with and without the addition of PEGylated phospholipids.

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BACKGROUND: Knowledge of the fatty acid composition of lipid classes in human plasma is an important factor in the investigation of human metabolism. Therefore, a method for the analysis of neutral lipid (NL), phospholipid (PL) and free fatty acids (FFA) in human plasma has been developed and validated. METHODS: Separation of lipid classes was carried out by solid phase extraction of the lipid extract. The fractions were transesterified and the resulting fatty acid methyl esters were determined by GC/FID. For the method to be validated, precision, detection and quantification limits, as well as recovery, were determined for combined lipid extraction, solid phase extraction and GC analysis. RESULTS: The lipid extraction was miniaturized and simplified by application of an ultrasound 'Sonotrode'. The resolution of lipid classes was optimized with appropriate standards added to a representative plasma sample. In addition, a rapid derivatization procedure using trimethylsulfoniumhydroxide was established. Low determination limits (1.5, 0.2 and 1.3 µg/g plasma for NL, PL and FFA, respectively) indicate that the method's sensitivity is sufficient to quantify even minor components. Furthermore, recovery for NL and PL fatty acids was found to range from 80% to 110%. The results were similar for FFA apart from more polar free fatty acids due to their higher solubility in water. Repetitive measurements showed very good precision apart from the long chain PUFA for which the coefficients of variation were significantly higher. CONCLUSIONS: The present method is applicable to the quantitation of fatty acids in lipid classes of human plasma including several minor components.

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EmrE is a multidrug transporter that utilises the proton gradient across bacterial cell membranes to pump hydrophobic cationic toxins out of the cell. The structure of EmrE is very unusual, because it is an asymmetric homodimer containing eight alpha-helices, six of which form the substrate-binding chamber and translocation pathway. Despite this structural information, the precise oligomeric order of EmrE in both the detergent-solubilised state and in vivo is unclear, although it must contain an even number of subunits to satisfy substrate-binding data. We have studied the oligomeric state of EmrE, purified in a functional form in dodecylmaltoside, by high-resolution size-exclusion chromatography (hrSEC) and by analytical ultracentrifugation. The data from equilibrium analytical ultracentrifugation were analysed using a measured density increment for the EmrE-lipid-detergent complex, which showed that the purified EmrE was predominantly a dimer. This value was consistent with the apparent mass for the EmrE-lipid-detergent complex (137 kDa) determined by hrSEC. EmrE was purified under different conditions using minimal concentrations of dodecylmaltoside, which would have maintained the structure of any putative higher oligomeric states: this EmrE preparation had an apparent mass of 206 kDa by hrSEC and equilibrium analytical ultracentrifugation showed unequivocally that EmrE was a dimer, although it was associated with a much larger mass of phospholipid. In addition, the effect of the substrate tetraphenylphosphonium on the oligomeric state was also analysed for both preparations of EmrE; velocity analytical ultracentrifugation showed that the substrate had no effect on the oligomeric state. Therefore, in the detergent dodecylmaltoside and under conditions where the protein is fully competent for substrate binding, EmrE is dimeric and there is no evidence from our data to suggest higher oligomeric states. These observations are discussed in relation to the recently published structures of EmrE from two- and three-dimensional crystals.

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Commercial activated partial thromboplastin time (APTT) reagents prepared with phospholipid extracted from animal or plant sources often differ in their response to heparin and coagulation factors and in their reference values. It is also known that there are variations in phospholipid composition and preparation procedure. The present study attempted to demonstrate that an APTT reagent based on synthetic phospholipids (phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine), which are substances of high purity and homogeneity, reduces batch-to-batch difference when compared with two traditional APTT reagents derived from rabbit brain and soybean. Three types of APTT reagent (SYN-APTT, RBT-APTT, SOY-APTT), prepared respectively from synthetic phospholipid, rabbit brain, and soybean, were tested. The total batch-to-batch difference was coefficient of variation (CV) 0.7-2.4% in the five reagents prepared from synthetic phospholipid (SYN-APTT), but CV 1.5-10.3% in the two traditional reagents (RBT-APTT and SOY-APTT). Additionally, high-performance liquid chromatography (HPLC) analysis showed clear variation in the phospholipid composition of the RBT-APTT and SOY-APTT reagents. In conclusion, the SYN-APTT reagent derived from synthetic phospholipid was shown to reduce batch-to-batch difference, and we therefore suggest that synthetic phospholipid is a substance useful in the preparation of APTT reagent and could contribute to stability of supply and uniform diagnosis.

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A phospholipid mixture extracted from cultured cells was directly analyzed by capillary (Cap) liquid chromatography (LC)/electrospray ionization (ESI) mass spectrometry (MS). Using a quadrupole mass spectrometer, we analyzed positive molecular ions, negative molecular ions, positive fragment ions and negative fragment ions under four different functions. In the analysis of the elution patterns of the phospholipids, a two-dimensional map, in which the first dimension is elution time and the second dimension is mass, proved useful. Consequently, four different maps can be obtained by each of four different functions. Among them, from negative fragment ions at high cone voltage in the negative ion mode, ions that originated from acyl fatty acid and phosphorylcholine, phosphorylethanolamine and cyclic inositol phosphate can be detected at specific elution times. The map from positive fragment ions at high cone voltage in the positive ion mode indicated ions such as diradylglycerol and derivatives of 1-alkyl or 1-alkenyl cyclic phosphatidic acid from phosphatidylethanolamine (PE), and phosphorylcholine from choline-containing phospholipids. The map produced from positive molecular ions indicated choline-containing phospholipids such as phosphatidylcholine, sphingomyelin, lysophosphatidylcholine and PE. The map of negative molecular ions effectively indicated acidic phospholipids such as phosphatidylinositol. We were able to obtain more than 500 molecular species of phospholipids by this method within a few hours immediately after extraction from culture cells using a mixture of chloroform and methanol (2:1). In this context, we concluded that the combination of Cap-LC and ESIMS seems to be very effective in the analysis of phospholipid classes and their molecular species.

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A practical, low cost capillary electrophoretic method for phospholipid products employed in pharmaceutical, cosmetic and food industries with quality control purposes is described. The phospholipid components of the samples were hydrolyzed prior to their electrophoretic separation. The carrier electrolyte to determine choline, ethanolamine and serine consisted of 5 mM imidazole, pH 3.0 using indirect UV detection at 214 nm. Inositol was quantified employing a buffer of 40 mM sodium borate, pH 10.0 at 40 degrees C and detection at 185 nm. Analytical parameters were evaluated and the results obtained of phospholipid composition in commercial samples from soybeans and animal sources showed acceptable values.

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This overview summarizes the ten randomized clinical trials that have compared different surfactant preparations. Six trials, enrolling 2,450 babies with respiratory distress syndrome (RDS), compared Survanta and Exosurf. Babies treated with the natural surfactant had lower oxygen requirements for at least 3 days than those treated with the synthetic surfactant. The babies treated with Survanta had lower risks of neonatal mortality (odds ratio, OR, 0.80; 95% confidence interval, CI, 0.65-1.00), retinopathy of prematurity (OR 0.68; 95% CI 0.50-0.94), and death or bronchopulmonary dysplasia (OR 0.84; 95% CI 0.70-1.00) when compared to those treated with Exosurf. Infasurf has been compared with Exosurf in two studies: one as prophylaxis and the other a rescue trial. Similar, although non-significant benefits were found for the natural surfactant. When all eight trials were included in a meta-analysis, there was a significant reduction in the odds of pulmonary air leaks (OR 0.52; 95% CI 0.41-0.66) for babies treated with natural as compared with synthetic surfactants. For seven trials (3,576 babies) comparing natural and synthetic surfactants to treat RDS (six comparing Survanta and Exosurf and one Infasurf and Exosurf), there was a significantly reduced risk of neonatal mortality (OR 0.80; 95% CI 0.66-0.97) with natural as compared with synthetic surfactant treatment. In two further trials different natural surfactant preparations have been compared. Reduced oxygen needs for 24 h after treatment were found for Infasurf and Curosurf, respectively, when compared to Survanta. Apparent longer-term benefits from these surfactants were not statistically significant. Further trials are needed to be certain of the differences between various surfactant preparations.

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To evaluate the thromboresistant properties of phospholipidic surface coatings mimicking the lipid surface of blood cells, we studied four different types of phospholipids bound onto PVC tubings in comparison to uncoated as well as heparin bonded controls. The samples analyzed included diacetylenic phospholipid coated as a monomeric treatment (A), diacetylenic phospholipid polymerised prior to being coated (B), and two types of polymeric phospholipids made using methacrylate containing monomers (C and D). A bovine (bodyweight 67 +/- 3 kg) left heart bypass model (pump flow 3.2 +/- 0.1 l/min) was selected and the surfaces were exposed to the blood stream up to 360 min without systemic heparinization. Thereafter another set of samples was exposed to stagnant blood over 20 min. Besides hemodynamic, hematologic and biochemical analyses, the macroscopic appearance of 119 blood exposed surface samples was graded semiquantitatively on a scale of 0 to 10: no macroscopic deposits = grade 0, 1 spot (1 mm diameter) = grade 1, 2 spots = grade 2, 5 or more spots = grade 5, up to 10% of the surface covered with clots = grade 6, 100% covered = grade 10 (P < 0.05 = *): mean grade of deposits was 0.0 +/- 0.0 for segments perfused and 0.0 +/- 0.0 for segments exposed to stagnant blood with surfaces exposing to the blood either heparin, phospholipid A, or phospholipid B (NS). Phospholipids C and D were graded 0.0 +/- 0.0 if perfused and 0.7 +/- 1.2 if exposed to stagnant blood.(ABSTRACT TRUNCATED AT 250 WORDS)

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